Full-length and truncated forms of vitronectin provide insight into effects of proteolytic processing on function

Gibson, Angelia D.; Peterson, Cynthia B.

doi:10.1016/s0167-4838(00)00290-9

Cited by 10 publications

(17 citation statements)

References 63 publications

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“…N-terminal sequencing confirmed the absence of the somatomedin B domain and cleavage of all but the final amino acid of the native signal sequence (valine). Gel-filtration chromatography of r⌬sBVN showed that it was oligomeric (data not shown), as observed previously with recombinant full-length and C-terminal truncated vitronectin expressed without a tag in the baculovirus system (41). The presence of the His 6 tag had no effect on oligomerization, because r⌬sBVN expressed without the tag was also oligomeric (data not shown).…”

Section: Resultssupporting

confidence: 84%

“…The first 40 amino acids of vitronectin were deleted, removing essentially all of the well structured disulfide cross-linked fold of the somatomedin B domain (typically delimited as residues 1-44), but retaining the RGD integrin-binding sequence found within a more flexible region at residues 45-47. The recombinant protein was produced using the baculovirus expression system as previously described (41). N-terminal sequencing confirmed the absence of the somatomedin B domain and cleavage of all but the final amino acid of the native signal sequence (valine).…”

Section: Resultsmentioning

confidence: 99%

“…Generation of Recombinant Vitronectin-Both full-length vitronectin and a deletion mutant of vitronectin lacking the first 40 amino acids were expressed in the baculovirus FastBac system (Invitrogen) as in the method described earlier (41). Both constructs were subcloned into the pFastBac-1 vector (Invitrogen) and utilized the endogenous vitronectin signal sequence to direct protein secretion.…”

Section: Methodsmentioning

confidence: 99%

“…Fractions containing protein were pooled, dialyzed, and concentrated. Protein concentration was determined as described previously (41) by quantitative ELISA and the Pierce BCA Protein Assay. The protein was also subjected to N-terminal protein sequencing, confirming the deletion of the somatomedin B domain as well as cleavage of all but the last amino acid (valine) of the signal sequence.…”

Section: Methodsmentioning

confidence: 99%

“…Heparin Binding Assays-A direct heparin binding assay was used to measure vitronectin binding to heparin-coated microtiter plates as previously described (41). Briefly, wells were coated with a 1 mg/ml solution of heparin (Sigma) then blocked using 3% casein in PBS.…”

Section: Methodsmentioning

confidence: 99%

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A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

Schar

Blouse

Minor

et al. 2008

Journal of Biological Chemistry

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Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (r⌬sBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins.

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

Schar

Blouse

Minor

et al. 2008

Journal of Biological Chemistry

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Model for the three‐dimensional structure of vitronectin: Predictions for the multi‐domain protein from threading and docking

Baburaj

Peterson

et al. 2001

Proteins

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The structure of vitronectin, an adhesive protein that circulates in high concentrations in human plasma, was predicted through a combination of computational methods and experimental approaches. Fold recognition and sequence-structure alignment were performed using the threading program PROSPECT for each of three structural domains, i.e., the N-terminal somatomedin B domain (residues 1-53), the central region that folds into a four-bladed beta-propeller domain (residues 131-342), and the C-terminal heparin-binding domain (residues 347-459). The atomic structure of each domain was generated using MODELLER, based on the alignment obtained from threading. Docking experiments between the central and C-terminal domains were conducted using the program GRAMM, with limits on the degrees of freedom from a known inter-domain disulfide bridge. The docked structure has a large inter-domain contact surface and defines a putative heparin-binding groove at the inter-domain interface. We also docked heparin together with the combined structure of the central and C-terminal domains, using GRAMM. The predictions from the threading and docking experiments are consistent with experimental data on purified plasma vitronectin pertaining to protease sensitivity, ligand-binding sites, and buried cysteines.

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Goat vitronectin: Characterization and binding to Staphylococcus aureus

Mahawar

Joshi

2008

Comparative Biochemistry and Physiology Part B: Biochemistry an

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Full-length and truncated forms of vitronectin provide insight into effects of proteolytic processing on function

Cited by 10 publications

References 63 publications

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

Model for the three‐dimensional structure of vitronectin: Predictions for the multi‐domain protein from threading and docking

Goat vitronectin: Characterization and binding to Staphylococcus aureus

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